RModBlock antisense oligonucleotides as a universal tool for precise and efficient inhibition of RNA modifications

The ability to selectively edit specific RNA modifications is needed to understand their roles in cellular function and disease. However, existing approaches, particularly catalytically inactive Cas (dCas)-based systems, have limited applicability owing to their reliance on eraser proteins, and their performance can vary depending on the modification type and target context. Here we introduce the RNA Modification-Blocking (RModBlock) strategy, which uses chemically modified antisense oligonucleotides (ASOs) with locked nucleic acid to precisely inhibit modifications at targeted sites. Most RNA modification writers, including those for m⁵C and pseudouridine, require specific structural contexts. Using representative modifications with and without eraser proteins, m⁵C and pseudouridine, respectively, we demonstrated that RModBlock ASOs blocked the formation of their structural context, inhibiting modified bases by up to 97% in human cells. RModBlock ASOs also inhibited m⁶A, despite its writer protein not requiring a strict structural context, suggesting broad applicability. Moreover, this strategy achieves comparable or superior performance when dCas13-eraser-based systems are applicable. Finally, by inhibiting cancer-relevant modifications and through in vivo delivery to the mouse liver, we highlight its therapeutic potential. This showcases the RModBlock strategy as a precise, efficient and versatile approach for manipulating RNA modifications, with broad applicability in basic and translational research.